Clathrin-mediated endocytosis (CME) is the major pathway for internalization from, and remodeling of the plasma membrane (PM) in mammalian cells. Thus, CME plays a fundamental role in all aspects of cell physiology, including nutrient uptake, signal transduction, cell motility, adhesion, polarity and differentiation. Consequently, defects in CME impinge on many human diseases, including cancer, cardiovascular disease, diabetes, neurological defects and others. CME is a multistep process initiated by the assembly of clathrin and the AP2 adaptor complex to form nascent CCPs. Subsequent steps including CCP stabilization, maturation and their scission from the PM to release clathrin coated vesicles are orchestrated by a myriad of endocytic accessory proteins (EAPs). Under the auspices of this grant, we have built an interdisciplinary team of biochemists, cell and molecular biologists, microscopists, engineers and computational biologists whose long- term goal is to define the mechanisms that regulate CME. Over the past 8 years, we have developed accurate and highly sensitive particle detection and tracking software and algorithms to quantitatively measure multiple orthogonal parameters relating to the dynamics of clathrin-eGFP labeled CCPs imaged by live cell total internal reflection fluorescence microscopy. We identified 3 kinetically-distinct subpopulations of CCPs, two short-lived abortive populations and longer-lived productive CCPs, as well as factors that differentially affect CCP initiation, stabilization and maturation. These data led us to propose that CCP maturation is gated during the first ~30s after initiation by an endocytic check-point that is, in part, regulated by the GTPase dynamin. Early recruitment of AP2-interacting EAPs to CCPs is also required for efficient curvature generation and to regulate multiple stages of the maturation process. The overarching goal of this proposal is to test this checkpoint hypothesis by identifying the determinants (i.e. individual or sets of EAPs) that function as potential sensors or effectors of the checkpoint, as well as those required for efficient CCP stabilization and maturation. To accomplish these goals, we propose three Specific Aims: 1) To measure the effect of individual and pairwise knockdown of EAPs on stages of CME through quantitative multi-parametric analyses of CCP dynamics; 2) To develop a molecularly explicit, mathematical model of the multi-step CCP maturation program and calibrate model parameters against measured CCP lifetime distributions in siRNA-treated cells, and 3) To, directly test, under minimally perturbing conditions, the functional assignments of EAPs predicted from the studies in Aim 1 and 2, and define the functional hierarchy of EAPs in regulating CCP maturation and progression beyond the endocytic checkpoint. These studies will provide unprecedented insights into the function of individual EAPs and their role in regulating key early stages of CCP stabilization of maturation. While most studies have focused on readily detectable early (CCP initiation) or late (CCV budding) events, we focus here on the less well understood stages of CCP stabilization and maturation that are central to the regulation of CME.